MODELING THE HEMOGLOBIN SWITCHPOINT WITH CYANOMET VALENCY HYBRIDS - RAMAN-SPECTROSCOPIC PROBES OF TERTIARY AND QUATERNARY STRUCTURE

Hybrid hemoglobins with cyanomet hemes in the alpha or in the beta chains have been investigated by resonance Raman (RR) spectroscopy, using ultraviolet (230 nm) and visible (441.6 nm) excitation. For the CO adducts, the UVRR spectra are identical with that of native HbCO, showing the tyrosine and tryptophan signals to be insensitive to ligand substitution within the R state. In the absence of CO, the doubly ligated hybrids show differences in the UVRR spectra, relative to the CO adducts, which are the superposition of two difference spectra: (1) the T-R difference spectrum obtained by subtracting the spectrum of HbCO from that of deoxyHb and (2) a perturbed R state spectrum, characteristic of deligated chains within the R state. These spectra arise from alterations, respectively, in the quaternary contacts of interface aromatic residues and in the tertiary contacts of interior aromatic residues. From the amplitudes of the difference spectra, the T state population was determined to be 30% for (alpha(FeII)beta(FeIII)CN)(2) and 43% for (alpha(FeIII)CN beta(FeII))(2), in good agreement with the kinetic analysis of Cassoly and Gibsoin [Cassoly, R., & Gibson, Q. H. (1972) J. Biol. Chem. 247, 7332]. Addition of inositol hexaphosphate (IHP) increased the T state population, but only by a modest amount, to 40 and 53%, respectively, in contrast to the frequent assumption that the T state conversion is quantitative in the presence of IHP. Since current understanding of the quaternary state dependence of the Fe-histidine stretching frequency is based on that assumption, the RR band envelope for this vibration was reexamined. For (alpha(FeII)beta(FeIII)CN)(2) th, band is readily resolvable into two components, whose areas yield the same T/R population ratios as do the UVRR difference spectra, in the presence and in the absence of IHP. The resolved components yield 223 and 210 cm(-1) as the R and T state frequencies; they both shift significantly upon IHP binding, to 219 and 203 cm(-1), revealing an IHP effect on tertiary as well as quaternary structure. For (alpha(FeIII)CN beta(FeII))(2), the components are not independently resolvable, but fixing their areas to correspond to the UVRR-derived T/R ratios yielded R and T state frequencies of 222 and 213 cm(-1), with no perceptible shift upon IHP binding. The T/R ratios are much lower than those derived from oxygenation curves for CN- adducts of partially oxidized Hb [Marden, M. C., Kiger, L., Kister, J., Bohn, B., & Poyart, C. (1991) Biophys. J. 60, 770]. This apparent discrepancy is ascribed to a preponderance of asymmetric hybrids (alpha(1)(FeII)beta(1)(FeIII)alpha(2)(FeIII)CN beta(2)(FeIII)CN), which Ackers et al. [Ackers, G. K., Doyle, M. L., Myers, D., & Daugherty, M. A. (1992) Science 255, 54] have shown to be thermodynamically more stable than the symmetric hybrids. The R state perturbation for unligated chains which is revealed in the UVRR spectra is believed to involve collapse of the E helix toward the heme and weakening of a tryptophan hydrogen bond between the E and A helices. This perturbation is suggested to account for the phenomenon of quaternary enhancement, an R state augmentation of ligand affinity for unligated chains.